In recent years, molecular diagnostics have gradually replaced or complemented culture-based, biochemical and immunological assays in routine microbiology laboratories. This book is the only lab manual focusing on this means of instruction , an approach particularly applicable to the microbiology laboratory. All the microbiological theory in the world means little if students cannot understand the Leboffe and Burton E.
Pierce is intended to act as a supplement to introductory microbiology laboratory manuals. Author : Michael J. This includes bacteria, archaea, viruses, fungi, prions, protozoa and algae, collectively known as 'microbes'.
Author : David O. Recent developments in sequencing technology allow researchers, and potentially practitioners, to examine microbial communities at unprecedented resolution and in multidisciplinary contexts. This detailed study of microbes facilitates the development of new forensic tools that use the structure and function of microbial communities as physical evidence.
Chapters cover: Experiment design Data analysis Sample preservation The influence of microbes on results from autopsy, toxicology, and histology Decomposition ecology Trace evidence This diverse, rapidly evolving field of study has the potential to provide high quality microbial evidence which can be replicated across laboratories, providing spatial and temporal evidence which could be crucial in a broad range of investigative contexts.
This book is intended as a resource for students, microbiologists, investigators, pathologists, and other forensic science professionals. Author : Richard J. With nothing more than basic lab equipment such as microscopes, Petri dishes, media, and a handful of reagents, you will learn to isolate, grow, and identify bacteria that live all around us.
This is no ordinary microbiology laboratory course; not only will you learn how to streak plates, use a microscope, perform a Gram stain, and prepare serial dilutions and spread plates—fundamental skills found in every microbiologist's toolkit—you will solve a series of public health—related challenges that many professional microbiologists encounter in their work. By the end of this course, you will: Determine the origin of a nosocomial infection. Using foundational and molecular methods, you will determine whether the infections occurring in hospitalized patients are the result of contaminated medical items.
Select the antibiotic to treat a patient with Crohn's disease. You will find minimum inhibitory concentrations of various antibiotics for a Pseudomonas strain associated with Crohn's disease. Pinpoint the source of lettuce contaminated with E. Using molecular tools you will investigate a common food safety challenge, antibiotic-resistant E. Find the farm releasing pathogens into a stream used for drinking water. Using bacteriophage load in water samples, you will locate the source of fecal contamination in the water supply of a village in an underdeveloped country.
Evaluate the potential of bacteria to cause a urinary tract infection. A culture is an in vitro technique of growing or cultivating microorganisms or only other cells in a suitable. Th e primary aim of constructing a culture. Culture media give artificial environment simulating natural conditions necessary. It is essential for existence of living cells. Th ey act as source of hydrogen and oxyg en. Golden granular hygroscopic powder which are o btained from meat, casein fibrin or soya be an flour.
It cont ains protein degradation products, carb ohydrates, i norganic salts, enzymes, excites and growth. It contains proteins, amino acid s, growth factors Vitamin B , Carbohydrates and inorganic salts like.
Functions: Source of growth factors and hence excellent stimulators of growth. It can be used as suitable. Dried mucilaginous substance obtained from gelidium species and other algae available as long shield or. Function: Act as source of energy, fermentation reactions are helpful in the identification and. No solidifying agents eg: agar is added while preparing the medium.
The most commonly used non-. Beef infusions are rich in minerals,. T hey are often supplemented with. The semi-solid medium remains in the semi-solid condition. It is prepared by adding small amount of agar. Agar is a complex carbohydrates prepared from algae like gelidium and gracillaria. Gelatin is an animal extract. When dissolved in water, it's in the form of a liquid. Many bacteria, when grown on a gelatin medium, produce a.
T his feature is important in the identification and. The sem i-solid m edium may be selective which promotes the growth of on e organism and re tards the. This type of medium can be used to study bacterial motility semisolid media. The solid medium is solid in consistency. It's used for colony characterization,. It's al so ca lled basal media. It consists of meat extract, peptone, Sodium Chloride and.
These have added ingredients for special purpose or for bringing out certain characteristics or providing. These m edia are prepared from pure chemical substances and the exact com position of the medium is. The nutritional requirements of som e microorganis ms include some additional ingredients o f unknown.
Chemical composition is not fully. No single medium or set of conditions can support the growth of all the different types of organisms that.
To cu ltivate, recognize, enumerate and isolate certain types of microorganisms many. In these media, substances like blood, serum or egg are added to a basal medium.
Enrichment Media. Some substances are added to liquid media with the result that wan ted organism grows more in number than. Te trathionate broth inhibit co liforms and allow typhoid paratyphoid bac illi to grow freely. It favors the growth of pa rticular microorganism.
T his is lik e enrichment media with the difference that. These media contain an indicator which chang es colour when a bacterium grows in them. Incorporation of sulphite in Wilson and Blair medium Salmonella typhi reduces sulphite to sulphide in. Wilson and Blair medium and the colonies o f S. Differential Media.
Media t hat distinguish between different groups of bacteria and even permit to identification of. A medium which has substances incorporated into. Such media are called differential media eg. Mac conkey medium consists of peptone, lactose, agar, neutral. It shows lactose fermenters, are colourless or pale this may also be termed indicator. Blood agar is an enriched medium, but also differentiates between hemolytic organisms and non- hemolytic. In peptone water al ong with appropriate indicator, a.
Delicate organisms l ike gonococci which may not survive the time taken which may not survive the time. The ingredients are dissolved in warm water and pH adjusted to 7.
Sabouraud's Dextrose Broth. Mueller — Hinton Agar. Methylene Blue Solution , Dissolved the methylene blue in distilled wa ter and was dispensed into regular staining bott les. Carbohydrate Fermentation. Mix all the ingredients, ex ce pt pheno l red indicator. Adjust pH to 7. Then add phenol red indicator. Dispense the medium in 8ml t est tubes containing the Durham's tubes. Sterilize the mediu m at 10lbs for Oxidation — Fermentation. Mix all the ingredients, expect Bromothymol blue indicator.
Then add Bromothymol blue. The medium is poured into the tube to a depth of about 4cm. Voges — Proskauer. Store in brown. Cool the volumetric flask in cold water with 80m l water, add KOH crystals, dissolve and make up toml. Citrate Utilization. MgSo 4 : 0. Dissolve the ingredients in ml di stilled water. Immediately before u se, mix equal volumes of. Di sodium phosphate : 1. Mono potassium phosphate : 0. Dissolve ingredients in ml distilled water.
Sterilize by autoclaving at 10lbs for 20 minutes. Sterilize the urea solution by autoclaving at 10lbs for 15 minutes,.
Dispense into sterilized test tubes and allow to set in a slanting. Mannitol Motility Test. Mix all the ingredients, expect phenol red indi cator. Triple Sugar Iron Agar Test. Mix all the ingredients, expect phenol red indicator. Then add phenol red ind icator. Allow the medium t o set in slopped form with a butt about 1 inch. This method was developed by two bacteriologists, Leoffler and Gaffkey in the laboratory of Robert Koch.
This method is routinely employed for the isolation of bacteria in p ure culture. In this method a sterilized. The process i s known as streaking and the plate so prepared is called a streak plate.
The m ain. A sterilized inoculating needle with a loop m ade up of either platinum or nichrome wire is used for. One loopful of specimen is transferred onto the surface of the agar plate in a sterile petridish and. This process is repeated thrice to streak out the. The first streak. The l ast streaks. The last streaks should thin ou t the culture sufficiently to give isolate colonies. Each colony usually represents the growth. Because of the high concentration of water in agar, some water of condensation for ms in petriplate during.
Moisture is likely to drip from t he cover to the surface of the agar and spread out, resulting in a. To avoid this, petriplates are routinely. Pure colonies can be obtained from well isolated colonies by transferring a small. The spread plate technique is used for the separation of a dilute, mixed population of the microorganisms so.
In this technique, a small volume of dilute microbial mixture is. The dispersed cells. Because the number of colonies will be equal to the number of viable.
In pour plate method, successive dilutions of the inoculum serially diluting the original specimen are. After incubation, the plates are.
The pure colonies may be isolated and transferred into. This technique is employed to estimate the viable bacterial. A culture that contains only one kind of microorganisms is called a pure culture. A culture which contains. Most of the cultures obtained in nature are.
Pure cultures are essential to study the cultural, morphological and physiological characters. There are different m ethods for obtaining pure cultures from m ixed cultures.
A single viable cell may be transferred on the culture medium to develop. This method is the standard practice for the isolation of tubercle. Bacteria with different optimum growth temperature can be separated by incubating at different. A mixture containing vegetative and spore.
In this method, the bacteria in th e vegetative. This method is useful for the isolation of tetanus bacilli from dust and similar. T his consists. The mixture is inoculated i nto the central. Microbiologist or laboratories concerned with microbial studies preserve cultures for a short period or many. These preserved cultures may be made available in. Some m ethods used for culture preservation include refrigeration, deep freezi ng, freezing under liquid.
Live cultures on a culture medium can be successfully stored in refrigerators or cold rooms maintained at. Generally, the metabolic activities of the microorganisms will be greatly slowed down at this. Thus growth will occur slowly, nutrients will be.
So subculturing of. In the case of bacteria, subculturing should be. In the case o f fungi, regular subculturing is necessary at intervals of 3 - 4. In t his method. The culture. Ampoules are removed from the mixture and placed directly into a. In this method, cell suspension in the presence of a stabilizing agent such as. Most species of bacteria can remain. The liquid nitrogen. Lyophilization or freeze drying is the rapid dehydration of organisms while they are in a frozen state.
Because metabolism. It is the most satisfactory method of long ter m preservation of microorganisms. It's universally used for the. Lyophilized cultures are revived by opening the vials adding. In simple staining, bacterial smear is stained with a single reagent, which produces a distinctive contrast.
A sim ple stain that stains the bacteria is the direct stain. On the basis of microscopic observation, bacteria appeared blue, violet and red respectively depending on. Differential staining requires the use of at least 3 chemical reagents that are applied sequentially to a heat.
Its function is to impart its colour to all cells. In order to establish a colour contrast, the second. B ased on the chemical composition of cellular components the.
The final reagent is the counter stain. Following discoloration, if the primary stai n is not washed out, the. If the primary stain is removed, the decolorized cellular components will accept and assume.
In this way, cell type or their structure can be distinguished from. On the basis of the stain that is retained the most important differential stain used in. The Gram stain, a differential stain was developed by Hans Christian Gram, a Danish physician , in Gram staining classifies bacteria into 2 major groups, Gram positive and Gram negative bacteria. The Gram. Gram positive cells have a thick peptidoglycan layer, whereas peptidoglycan layer in Gram negative cells is.
In Gram negative, the higher amount of lipid in the formation of large pores thus facilitating the leakage of. In contrast, the Gram positive cell wall are thick and composed mainly of proteins. The bacteria which retain the. Gram positive, where as those that lose the crystal violet used counter stain, saffranin appear red are called. The Gram stain uses different reagents in the order, crystal violet, iodine solution, alcohol and saffranin. Those bacteria that appear blue are referred to as Gram positive and these appearing pink are described as.
It may be dipped i n alcohol and polished dr y with tissue, or. Place a thin film of. Using good aseptic technique,. Be certain the loop. Close and return the tube to the rack. Sterilize the loop. Now turn the slide over. You should have. Start your examination with the low -power. It is helpful to focus first on one edge of the drop, which will appear as a.
The light sho uld be reduced with the iris diaphragm and, if nec essary, by lowering the. You should be able to focus easily on the yeast cells in the suspension. If you have trouble. Although the yeast cells will be obvious becau se of their larger. Fam ily : Enterobacteriaceace. Genus : Escherichia. Species : coli. Small, regular, circular, lactose fermenting colonies.
Table 1. Biochemical tests of E. Genus : Pseudomonas. Staphylococcus aureus. Family : Staphylococcaceae. Genus : Staphylococcus. Species : aur eus. Small, regular, circular, entire, smooth, conv ex, opaque, lactose fermenting colonies.
To determine the ability of microorganisms to degrade and ferment carbohydrate with the production of. Most microorganisms use carbohydrate differently depending on their enzymes components. The pH indicator Phenol Red is used to detect the. Thi s. In some cases, acid production is accompanied by the evaluation of gas such as H ydrogen or Carbo n. To detect the presence of gas produced or Durham's tube an inverted inner vial is placed in the.
Cultures that are not capable of fermenting any carbohydrate and not producing concomitant evolution of. All carbohydrate broth cultures were observed for colour and presence or absence of gas b ubble by. A — Only acid is formed; the broth has turned yellow. Table 5. Carbohydrate — Fermentation Test. Fermented with aci d. This method depends upon the use of semisolid tube. If acid is produced t hroughout the. Fermenting or ganism Enterobacteriaceace, Vibrio.
Oxidizing organisms Pseudomonas produce an acidic. Tryptophan an essentia l amino acid oxidized by so me bacteria. In this experiment, the. This ability to hydroly se tryptophan with the production of indole is not a characteristic of all. The presence of i ndole is detected by adding. Kovac's reagent, which produces a cherry red reagent layers. This colour is produced by the reagent which.
Culture producing a red re agent l ayers following addition of the Kovac's reagent are indole positive. Another reagent used is Ehrlisch's reagent.
It's believed to be more sensitive than Kovac's reagent and is. Development of cherry red colour in the to p layer of the tube is a positive test. Ab sence of red colouration is. Positive: E. To determine the ability of microorganism to oxidize glucose with. All enteric organisms oxidize glucose for energy production an d. In this test, the pH. The test can be used in differ en tiating Escherichia coli.
The low acid end products produce acidic pH 4 which is stabilized and. During the later incubation period Enterobacter aerogenes. At a pH of 4, Methyl red indicator will t urn red throughout the t ube, which is indicating of a positive test. At pH 6, still indicating the presence of acid but with a lower hydrogen ion concentration, the indicators. The colour of MR reagents remaining red is a pos itive test and the colour turning to yellow is negat ive.
MR positive — E. MR negative — Klebsiella, Enterobacter sp. To determine the ability of many microorganism s to produce acetone acetyl methyl carbino l during. Detection o f the a cetyl m ethyl carbinol requires this end p roduct to be. At a result, a pink complex a guanidine group that is present. As a result, a pink complex i s complex is formed imparting a rose colou r to the medium.
Development of deep rose colour in culture with in a minute following the addition of Barrett's reagent is. The absence of rose. Note : the colour may be more intense at the sur face. Red colour formation indicates a positive test and colour change is negative. Positive — Klebsiella sp. Negative — E. To determine the ability of a microorganism to utilize citrate as the sole source of carbo n and as energy. In the absence of glucose or lactose some microorganisms utilize citrate as.
This ability depends on the presence of citrase enzyme that facilit at es the transport of. Citrate, the first major intermediate i n Krebs's cycle is produced by th e condensation of. These products are then enzym atically converted to. During this reaction t he. This changes the. Bromothymol blue i s green when acidic pH 6. When alkaline pH 7. Formation of blue colour. Colour of the medium if t urned blue, a positive result is indicated. Colour of the medium remains as green,.
The reduction of nitrate by some aerobic and facultative anaerobic microorganisms occur in t he absence of. Some organisms possess the enzymatic capac ity to act further on nitrates to reduce them to ammonia or. Nitrate reduction can be determ ined by cultivating organisms a nitrate broth m edium. The medium is. The semisolid impedes the. An organisms ability to reduce nitrate to nitrite is determined by the addition of two reagent. This test determines the production of an enzyme called nitrate reductase, resulting in the reduction of.
With this enzyme, nitrate is reduced to nitrite NO 2. It then forms nitrous acid that r eacts. The development of red colour , therefore, verifies that nitrates were not reduced to nitrites by the. If nitrites were reduced a negative nitrate reduction had occurred. If the addition of zinc does not. This is a positive reaction or result.
Reduction of nitrate is generally an anaerobic respiration in which an. Bacterial broth, Nitrate broth, Nitrate reag ent and inoculation loop. The tubes were observed to see a red colour has. A minute quant ity of zinc was adde d to cultures. Development of red colour indicates nitra te positive and no colour change indicates a negative test.
Eg: Positive: all members of Enterobacteriacea ce. Negative: Haemophilus duceryi. Urease is a hydrolytic enzyme that attacks nitrogen and. The presence of u rease is. As the. This i s a positive reaction for the presence of. Failure of deep pink colour to develop is evidence of. Bacteri al broth cultures, Christener's urea agar slant and the inoculation loo p.
Eg Urease Positive — Klebsiella sp. Urease Negative — E. To detect whether the given organism is motile and also mannitol is fermenting or not. Mannitol motility test medium is an example of semisolid agar media; motile bacteria swarm and give a. The final sterile medium should be. After incubating the stabbed culture, non-motile bacteria generally give growth.
Motile bacteria typically g ive diffused, hazy growth that spread s throughout the medium. This test also helps to identify whether the microorganisms fer ment Mannitol. It produces acidic end products which in turn change the red colour of phenol red i ndicator to. Bacterial culture broth, mannitol fermentation m edia semisolid and inoculation loop.
Diffused growth — Motile bacteria eg:. To identify the microorganisms based on the ability to ferment the carbohydrates Glucose, Sucrose and. The triple sugar- iron agar test is de signed to differentiate among the different g roups or genera of the. Enterobacteriaceace, which are all Gram negative bacilli capable of ferm enting glucose with the production. This differentiation is based on. Carbohydrate fermentation is i ndicated by the presence of gas and a visible colou r.
The production of hydrogen sulphide in the medium is indicated by. To facilitate the observation of carbohy drate utiliza tion patterns, TSI Agar contains three f ermentative. Due to the production. The acid base indicator Phenol red is incorporated for detecting. This is indicated by the. Sodium thiosulfate. Posting Komentar. Information Receive in your inbox the latest content and participate in the promotions and benefits we have prepared for you. Newslater Receive in your inbox the latest content and participate in the promotions and benefits we have prepared for you.
0コメント